Sowa et al. Mol Brain (2021) 14:57 https://doi.org/10.1186/s13041-020-00723-0

RESEARCH Open Access Neurodegenerative phosphoprotein signaling landscape in models of SCA3 Anna S. Sowa1,2 , Taissia G. Popova3, Tina Harmuth1,2 , Jonasz J. Weber1,2,4 , Priscila Pereira Sena1,2 , Jana Schmidt1,2 , Jeannette Hübener‑Schmid1,2 and Thorsten Schmidt1,2*

Abstract Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disorder resulting from an aberrant expansion of a polyglutamine stretch in the ataxin-3 protein and subsequent neuronal death. The underlying intracellular signaling pathways are currently unknown. We applied the Reverse-phase Protein MicroArray (RPMA) technology to assess the levels of 50 signaling proteins (in phosphorylated and total forms) using three in vitro and in vivo models expressing expanded ataxin-3: (i) human embryonic kidney (HEK293T) cells stably transfected with human ataxin-3 constructs, (ii) mouse embryonic fbroblasts (MEF) from SCA3 transgenic mice, and (iii) whole brains from SCA3 transgenic mice. All three models demonstrated a high degree of similarity sharing a subset of phosphorylated proteins involved in the PI3K/AKT/GSK3/mTOR pathway. Expanded ataxin-3 strongly interfered (by stimulation or suppression) with normal ataxin-3 signaling consistent with the pathogenic role of the polyglutamine expansion. In comparison with normal ataxin-3, expanded ataxin-3 caused a pro-survival stimulation of the ERK pathway along with reduced pro-apoptotic and transcriptional responses. Keywords: Spinocerebellar ataxia type 3 (SCA3), Machado-Joseph disease (MJD), Ataxin-3 (ATXN3), RPMA, Neurodegeneration, pERK, AKT (PKB), mTOR, Phosphoprotein

Introduction pathways of neurodegeneration remain elusive. Protein Spinocerebellar ataxia type 3 (SCA3), also known as activation mapping and identifcation of critical nodes Machado-Joseph disease (MJD), is an autosomal domi- in the signaling network represent central approaches nantly inherited ataxia which is characterized by defcits required to elucidate the underlying nature of the dis- in gait, movement, and coordination linked to a CAG ease and for the development of efective therapeutic repeat expansion in the ATXN3 gene and a concordant interventions. Traditional low-throughput methods of polyglutamine expansion in the ataxin-3 protein. Tere- protein analysis such as Western blotting and immu- fore, SCA3 falls into the group of polyglutamine disor- nohistochemistry have served an important role in ders which includes Huntington’s disease and within understanding SCA3, but their well-known limita- the broader category of neurodegenerative disorders to tions include difculty in analyzing complex signaling include Parkinson’s disease and Alzheimer’s disease [55]. networks at the level of protein expression and post- Although the gene responsible for SCA3 was discov- translational modifcation. On the other hand, DNA ered more than 25 years ago [25], the cellular signaling microarrays and RNA sequencing provide readouts restricted to the mRNA level with little follow-up on how these changes manifest at the protein level. While *Correspondence: [email protected] RNA encodes information about cellular status, pro- 1 Institute of Medical Genetics and Applied Genomics, University teins are the ultimate drivers of the cellular machin- of Tuebingen, Calwerstrasse 7, 72076 Tuebingen, Germany Full list of author information is available at the end of the article ery serving in key mechanism of cell signaling through

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post-translational modifcations. Reversible protein described in Schmidt et al. [44]. Mice were sacrifced at phosphorylation, especially on serine, threonine or 12 months of age. tyrosine residues, is one of the most important and well-studied post-translational regulating protein func- RPMA analysis tion and signal transmission [5]. However, phosphoryl- For Reverse-phase Protein MicroArray Analysis (RPMA, ated proteins are notoriously difcult to analyze on a [60]) we used an established and pre-validated set of 50 mass scale by traditional methods such as Western blot diferent antibodies [38, 39] directed against the total and especially when small amounts of samples need to be phosphorylated forms of signaling proteins which cover a tested with high sensitivity. wide array of signaling pathways relevant to the cell fate To that end we utilized the Reverse-phase Protein including apoptosis, survival, and autophagy (Additional MicroArray (RPMA) technology, a sensitive, quantitative, fle 1: Tables S1 and S2). All antibodies were validated and high-throughput immunoassay to analyze protein for specifcity prior to testing. RPMA analysis was per- and post-translational modifcation at the level of total formed according to Einspahr et al. [17]. Samples were protein abundance or specifc phosphorylation. In the printed onto the array slides at concentrations 100%, RPMA analysis a few microliters of cell lysates are printed 50%, 25%, and 12.5% in duplicates and prepared for stain- onto nitrocellulose slides which are then probed with the ing by treating with Reblot (Chemicon, CA). Slides were protein-specifc antibodies. Te amount of bound anti- treated with blocking solution I-block (Applied Biosys- body is quantitated using a highly sensitive colorimetric tems, MA) at 2 g/l and 0.5% Tween-20 in PBS. Te total or fuorometric procedure [60]. protein amounts loaded on the chip were estimated using Here, we present an RPMA-based analysis of three SYPRO Ruby Protein Blot Stain (Invitrogen, CA) accord- SCA3 models including human embryonic kidney ing to the manufacturer’s instructions and imaged on the (HEK293T) cells stably-transfected with plasmids encod- NovaRay scanner (Alpha Innotech, CA). Blocked arrays ing diferent ataxin-3 variants with normal (15 glu- were stained with antibodies on an automated slide tamines, ­HEK15Q) and expanded polyglutamine repeat stainer (Dako, CA) using the Catalyzed Signal Ampli- (148 glutamines, ­HEK148Q), mouse embryonic fbroblasts fcation System kit according to the manufacturer’s rec- (MEF) from ataxin-3 148Q ­(MEF148Q, [12]) and ataxin-3 ommendation (Dako, CA). A signal was generated using knockout mice (MEF­ KO, [44]), and whole-brain samples streptavidin-conjugated IRDye 680 (LI-COR Biosciences, from a SCA3 mouse model (CamKII/SCA377Q, [11, 43, NE). Stained slides were scanned on the NovaRay scan- 48]). Our data provide assessment of the phosphoprotein ner. Te TIF images of the antibody and SYPRO-stained signaling landscape contributing to the development of slides were analyzed using MicroVigene v2.9.9.9 software the disease phenotype in the context of diferent SCA3 (VigeneTech, MA). Briefy, MicroVigene performed spot models and suggest that the RPMA technology can be fnding, local background subtraction (using local and broadly applied to characterize neurodegenerative disor- slide average intensity), replicate averaging and total ders thus assisting in biomarker identifcation and devel- protein normalization, producing a single value for each oping novel targets for therapy. sample at each dilution. All signal values produced for data analysis were at least two standard deviations above background. All four dilutions of each sample were ana- Materials and methods lyzed for linear regression and only measurements which Cell and Mouse models of SCA3 met linearity were kept for analysis. For each sample and HEK293T cells were stably transfected with a plas- antibody, SCA3 samples were normalized to the cor- mid expressing green fuorescent protein (GFP)-tagged responding protein concentration in the wild-type sam- ataxin-3 with 15 glutamines ­(HEK15Q), ataxin-3 with 148 ple at the same dilution and averaged across all available glutamines ­(HEK148Q), or empty GFP plasmid ­(HEKempty) dilutions. Tese were then analyzed by the t-test to deter- as control. Transfected cells were sorted by FACS and mine the p-value for each averaged protein level. All dif- highly expressing cells were collected for analysis [48]. ferences compared to wild-type found signifcant with Mouse embryonic fbroblasts (MEF) were isolated as 95% confdence are shown in Additional fle 1: Table S1. described in Hübener et al. [24] from the ataxin-3 148Q ­(MEF148Q), ataxin-3 knockout (MEF­ KO), and wild-type mice ­(MEFwt). Te ataxin-3 knockout mouse line is Western blot analysis described in Schmitt et al. [45] and the ataxin-3 148Q Western blot analyses were essentially performed as mouse line was generated as described in Boy et al. described previously [48, 56] with 30 µg of protein loaded [12]. C57BL/6 wild-type mice were used to isolate con- from each sample on Tris–glycine or Bis–Tris SDS trol fbroblasts. Te CamKII/SCA377Q mouse model is polyacrylamide gels and separated electrophoretically. Sowa et al. Mol Brain (2021) 14:57 Page 3 of 10

Afterwards, proteins were transferred to 0.2 µm nitrocel- response to expanded ataxin-3 were found to be rather lulose membranes (GE Healthcare, Dornstadt, Germany) similar (Fig. 1a). Pairwise comparison of the HEK­ 148Q and using the respective transfer bufer. After blocking, the ­MEF148Q cell responses in spite of the diferent species membranes were incubated with the same primary anti- origin (human vs. mouse) revealed only four proteins as bodies as used in the RPMA analysis (for details and dilu- non-shared low responders (with levels changed by less tions see Additional fle 1: Table S2) diluted in TBST at than ± 0.1). As expected, brain of the SCA3 transgenic 4 °C overnight. Following the incubation with a fuores- mouse model was more distant from the cultured cells: cence- or HRP-labeled secondary antibody, membranes 16 responses were found unique for the MEF­ 148Q cells were detected using an Odyssey Fc Imager (LI-COR Bio- or the analyzed brains. All three models shared signal- sciences, Bad Homburg, Germany) and quantifed using ing nodes of major cell pathways important for cell fate the Image Studio Software (LI-COR) or ImageJ. Statis- (Table 1). A fragment of the signaling network illustrat- tical analyses were performed using GraphPad Prism ing these results is shown in Fig. 1b. Comparison of the 8.40 for Windows (GraphPad Software, San Diego, CA). ­HEK15Q and HEK­ 148Q cells identifed changes due to Data is shown as arithmetic means ± SEM. Outliers were the polyglutamine expansion within ataxin-3 (Addi- identifed using Grubbs’ test with alpha = 0.05. Statistical tional fle 1: Table S1). Among the prominent diferences signifcance of data sets was determined using Student’s (above the ± 20% threshold) between these cells the poly- t-test and p-values less than or equal to 0.05 were consid- glutamine expansion caused reduction of the levels of ered as statistically signifcant. apoptotic and transcriptional regulators (Table 1). Te increased responses included the pro-survival ERK- Results p70S6K pathway (Table 1). Tere is mounting evidence that infammatory signaling cascades are involved in the development and patho- Efect of wild‑type ataxin‑3 and the polyglutamine genesis of SCA3 [4, 15]. We thus generated a snapshot expansion of the SCA3 degenerative landscape via a large screen of To evaluate the contribution of normal (non- phosphorylated signaling proteins which act as principal expanded) ataxin-3 in the cell signaling we analyzed regulators of signal transduction across multiple signal- cells lacking ataxin-3 ­(MEFKO) in comparison with ing networks. Te list of tested proteins and their levels wild-type cells (MEF­ wt). The proteins were grouped relative to corresponding controls are presented in Addi- together based on their stimulation or suppression tional fle 1: Table S1. relative to the wild-type cells (Fig. 2). It is typically Our results show that the overexpression of both nor- assumed that the proteins in the knock-out (KO) cells mal and expanded ataxin-3 has a profound efect on would display opposite effects (suppression vs. stimu- several signaling pathways. Of signifcant note is the lation) compared to wild-type cells. We therefore phosphatidyl inositol 3-phosphate kinase (PI3K) cell compared the reaction towards the loss of ataxin-3 survival pathway. Phosphorylation of its key members ­(MEFKO vs. ­MEFwt) with the reaction induced by the (Table 1) is robustly altered. polyglutamine expansion within ataxin-3 ­(MEF148Q vs. ­MEFwt). Figure 2 shows that a number of responses Major signaling responses altered by expanded ataxin‑3 induced by the presence of expanded ataxin-3 were in SCA3 models suppressed by the presence of wild-type ataxin-3 Although we applied a wide range of cellular and ani- (stimulated in ­MEFKO cell). On the other hand, a mal models, the sets of altered signaling proteins in large group of responses including the major cell fate

Table 1 Major pathways altered in SCA3 models Pathway Altered members

PI3K cell survival pathway AKT, PTEN, p70S6K, GSK3, RPS6 Cell fate pathways AKT, PTEN, JNK, ERK, GSK3, Src, p53, eNOS, p70S6K, RPS6, caspases 3, caspase 7, caspase 9 Apoptotic regulation Bax, Bcl-xL, XIAP Transcriptional regulation STAT1, STAT3, IκBα Pro-survival ERK-p70S6K pathway ERK, p70S6K, RPS6 AKT/mTOR pathway targets ERK, eNOS, GSK3, p53, p70S6K, PTEN, RPS6, Src

The table sums up the respective members of major pathways and pathway targets found to be altered in our RPMA analysis of SCA3 models Sowa et al. Mol Brain (2021) 14:57 Page 4 of 10

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Fig. 1 Altered signaling proteins in models of SCA3. a Signaling proteins responding to expanded ataxin-3 expression in the indicated SCA3 models ­(HEK148Q, ­MEF148Q and brain of SCA3 mouse model) in comparison to the respective controls (HEK­ wt, ­MEFwt, and control brain). In all three diferent SCA3 disease models, we observed a signifcant overlap of dysregulated signaling proteins. For clarity, only the proteins displaying alterations of more that 20% relative to the corresponding control levels are shown. b A fragment of the signaling network illustrating ± connections between shared proteins in a proteins were stimulated by the presence of wild-type efects on the mTOR pathway by the expression of ataxin-3 (suppressed in ­MEFKO cells) but suppressed expanded ataxin-3 [21,23]. in cells expressing expanded ataxin-3. No responses stimulated by wild-type ataxin-3 were found to be fur- Discussion ther amplified by the presence of expanded ataxin-3. Polyglutamine expanded ataxin‑3 decreases pro‑survival As expected, the SCA3 whole brain samples dem- signaling via the AKT pathway onstrated a certain degree of specificity compared to Te serine/threonine-protein kinase AKT is a key pro- the ­MEF148Q cells (Fig. 2). In spite of these specifics tein kinase involved in survival and growth with physi- the results from both models demonstrated that the ological links to neurological disorders. Te levels of expanded ataxin-3 strongly interfered with the normal Serine 473-phosphorylated AKT (p-AKTS473), repre- ataxin-3 signaling. senting an active form of this protein, were decreased To corroborate the results of the RPMA analy- in all three models indicating a drop in pro-survival sis, we validated a selection of marker proteins found signaling due to the 148Q expansion. AKT signal- dysregulated in the mouse brain samples by West- ing cascades include a number of proteins previously ern blot, demonstrating a strong consistency with our implicated in SCA3 such as GSK3, FOXO and mTOR high-throughput approach (Fig. 3a, b). Tis includes [4, 18, 27, 42]. An increased susceptibility towards oxi- the downregulation of the phosphorylated forms of dative stress (via FOXO4 and SOD2) was suggested AKT, ERK, GSK3 and JNK. To further dissect the to be a potential contributor to neuronal cell death apparently downregulated AKT/mTOR pathway, we in SCA3 and AKT, ERK, and JNK are all among the investigated additional proteins namely total and phos- known kinases which target FOXOs and contribute to phorylated levels of mTOR itself and its upstream their activation and localization [4, 27]. inhibitor AMPKβ1. Western blot analysis showed a Upstream of AKT lies the PI3K pathway. Tis path- trend towards a reduction of the AKT and p70S6K- way is balanced and activated by the tumor suppressor mediated activating phosphorylation of mTOR at phosphatase and tensin homolog PTEN. In our analyses, Ser2448, and signifcantly increased levels of pSer108- we observed an increase of both the total and phospho- AMPKb1 (Fig. 3c, d), both confrming rather inhibitory rylated levels of PTEN in SCA3 mouse brain samples Sowa et al. Mol Brain (2021) 14:57 Page 5 of 10

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Fig. 2 Side-by side comparison of signaling in reaction to the presence of wild-type and expanded ataxin-3 in MEF cells (a) and SCA3 mouse brain (b). Shown are the relative protein levels of signaling proteins altered upon the presence of wild-type ataxin-3 (comparison of MEF­ KO with ­MEFwt) or expanded ataxin-3 (MEF­ 148Q vs. MEF­ wt) in MEF cells (a) as well as in mouse brain (SCA3 vs. wt mouse brain, b). Proteins are grouped according their stimulation ( ) / suppression ( ) by wild-type (ataxin-3wt) and expanded ataxin-3 (ataxin-3exp). For clarity, only the protein pairs displaying ↑ ↓ alterations of more that 20% in one or both proteins relative to the corresponding control levels are shown ± and a decrease in HEK293T and MEF cells expressing [30]. With respect to SCA3 it is known to phosphoryl- expanded ataxin-3. Interestingly, ataxin-3 is suggested to ate ataxin-3 and thus regulate its aggregation propensity be responsible for repressing PTEN transcription in can- [19, 37]. Furthermore, the phosphorylation of ataxin-3 cer [42] which further emphasizes the importance of the by GSK3 contributes to its nuclear translocation [37], a AKT signaling pathway in disease progression. process increasing the toxicity of expanded ataxin-3 and required for the manifestation of the disease [9, 48]. Te Altered GSK3 signaling in SCA3 models reduction of GSK3 may therefore refect a protective Importantly, we also saw a reduction of phosphoryl- mechanism against this increase of toxicity. ated glycogen synthase kinase 3 (GSK3) shared among GSK3 was also the frst reported substrate of AKT and all SCA3 models investigated. GSK3α and GSK3β are the two are functionally similar. GSK3 is a strong pro- constitutively active, proline-directed serine/threonine moter of Toll-like receptors-induced production of pro- kinases involved in a variety of cellular processes includ- infammatory cytokines such as IL-6 which was found to ing glycogen metabolism [58], gene transcription [50], be altered in SCA3 patient samples [18]. AKT is known apoptosis [51] and microtubule stability [3, 13]. GSK3 to exert an inhibitory role on GSK3 via phosphorylation is known to play a role in several neurological disorders of Ser21/Ser9 (p-GSK3S21/S9) [22]. Consistent with the Sowa et al. Mol Brain (2021) 14:57 Page 6 of 10

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Fig. 3 Confrmation of RPMA data by Western blot analysis. For validation of RPMA data, Western blot analyses of mouse brains of SCA3 and wild-type mice were performed for selected targets. The same antibodies as in the RPMA analysis were used. Blot images (a) and graphs (b) showing their quantifcation. Quantifed bands are marked with a black arrow. β-actin was used as loading control. In the SCA3 mouse model, transgenic expanded ataxin-3 (tg) is detected in addition to the endogenous ataxin-3 (eg). In addition, total and phosphorylated levels of AMPKβ1 and mTOR were analyzed (c and d) to further dissect the impact of expanded ataxin-3 on the mTOR signaling pathway. The bars correspond to means (wt n 3, SCA3 n 2–4) SEM. Statistical signifcant diferences (t-test) are marked (*, p < 0.05; **, p < 0.01) = = ± Sowa et al. Mol Brain (2021) 14:57 Page 7 of 10

observed decrease in p-AKTS473, we detected a reduced the calcium-dependent phosphorylation of ataxin-1 [26] inhibitory phosphorylation of GSK3 in HEK293T and and the downregulation of ERK reduces levels of ataxin-1 MEF cells expressing expanded ataxin-3. and suppresses neurodegeneration in Drosophila and Furthermore, GSK3 is required for activation of STAT mice [20]. ERK is further able to act on downstream path- proteins in astrocytes, microglia and macrophages [8]. ways of AKT and mTOR via control of the p70S6 kinase Accordingly, our SCA3 cell models showed changes and the S6 ribosomal subunit [34]. Te phosphorylated in seven proteins compared to two STAT proteins in ERK (p-ERK1/2T202/Y204) was reduced in all models tested the brain samples, while ­MEFKO cells showed profound including the knockout ­MEFKO cells. Te latter suggested alterations of STAT levels in response to the absence of that the stimulating efect of wild-type ataxin-3 in regu- ataxin-3 (Additional fle 1: Table S1 and Fig. 2). Tese lating p-ERK1/2T202/Y204 levels was abrogated by the poly- observations reinforce the link between ataxin-3 and glutamine expansion. We also found that similar to ERK, GSK3 and should prompt the feld to consider the role another MAPK, stress-activated p-JNKT183/Y185 displayed of insulin and glucose in brain homeostasis and neuronal a strong inhibition by the expanded ataxin-3. degeneration as a metabolic syndrome. It is noteworthy, An additional target of AKT is the endothelial nitric that GSK3 inhibitors recently gained attention in terms oxide synthase (eNOS) which catalyzes the production of of their potential for diabetes, cancer, and neurodegener- nitric oxide. We observed a decrease in eNOS activation ation [33] highlighting a therapeutic potential for SCA3. by ataxin-3 148Q in all models similar to ERK. Te phos- phorylation of eNOS at Ser1177 (p-eNOSS1177) by AKT SCA3 models exhibit alterations of AKT‑associated factors activates this enzyme. In terms of neurodegeneration, Tumor suppressor protein p53 is considered the “guard- alterations in eNOS have been linked to blood–brain ian of the genome” [28] because of its important role in barrier integrity [6] which directly points to the impor- determining cell fate. A tight regulation of p53 is criti- tance of peripheral infammation in propagation of CNS cal in maintaining homeostasis and keeping a balance dysfunction. between its cancer-suppressive and age-promoting func- tions [10]. Te interaction between p53 and GSK3 is Indication of impaired mTOR activation in SCA3 models hypothesized to be involved in aggregate clearance and P70S6 kinase is a mitogen-activated kinase afected by neurodegeneration: the Alzheimer’s disease hypothesis ERK signaling downstream of mTOR and AKT. It phos- suggests that under normal conditions, GSK3 is involved phorylates the S6 protein of the 40S ribosomal subunit in keeping p53 levels low while under conditions of cellu- (RPS6) and thus controls mRNA translation. Te phos- lar stress (e.g. aggregation) p53 is stabilized, proteasomal phorylation of RPS6 is commonly used as a readout function declines, and levels of p53 increase [40, 51]. We of mTOR activation [36] and other work suggests that observed that the levels of activated p-p53S15 increased in the state of Ser240/Ser244 phosphorylation of RPS6 ­MEF148Q cells and in SCA3 mouse brains in contrast to (p-RPS6S240/S244) can be used to estimate the neuronal its suppression by wild-type ataxin-3 (increase in MEF­ KO activity state of striatal cholinergic interneurons [7]. We cells). Recently, p53 was identifed as a substrate of wild- observed decreased levels of p-RPS6S240/S244 in all models type ataxin-3 and the polyglutamine expansion was asso- and ­MEFKO cells indicating a negative impact of the pres- ciated to increased p53-dependent neuronal death [31]. ence of expanded ataxin-3 on mTOR activation. Moreo- One can hypothesize that SCA3 and other neurodegen- ver, an interaction between ataxin-3 and the kinase erative disorders involve a positive feedback between (ribosomal protein S6 kinase alpha-1) phosphorylating proteasomal degradation, aggregation, GSK3 activa- RPS6 has also been suggested [1, 52]. tion, and increased p53 levels. Besides p53, the strongest Along with p70S6K, another important neuron-related increase in MEF cells both as reaction to the expression kinase connected with mTOR is Src. Our data indicate of expanded ataxin-3 and to the loss of ataxin-3 were an increased activity of Src in our models: Te Tyr527 observed for HSP90 and SUMO 2/3. Both proteins were phosphorylation site of Src (p-SrcY527) which renders the implicated before in SCA3 [2, 47] and our results fur- enzyme less active was decreased in HEK­ 148Q, ­MEF148Q ther stress their relevance. Ataxin-3 is SUMOylated and and mouse brain samples. Although frst identifed as a the SUMOylation of ataxin-3 regulates its function [2]. proto-oncogene, Src was found to be expressed in dif- Moreover, the inhibition of HSP90 turned out as promis- ferentiated, post-mitotic neurons and is important for ing therapeutic strategy in a mouse model for SCA3 [47]. neuronal diferentiation and neurite outgrowth with Te extracellular signal regulated kinase ERK is a mem- the capability to control ion channel activity and syn- ber of the MAPK pathway. It is a key protein lying at the aptic transmission [43]. Src is involved in the intracel- intersection of proliferation, diferentiation, and survival lular release of calcium stores from the endoplasmic [29]. It was previously shown that ERK is responsible for reticulum [53]. Concordant with our data, a reduced Sowa et al. Mol Brain (2021) 14:57 Page 8 of 10

phophorylation of Src has also been observed in a dif- of survival or death outcomes cannot be predicted with ferent mouse model of SCA3 [59]. Moreover, Src has certainty based on our data only. Nevertheless, it seems been linked to Huntington’s disease where expression of reasonable to conclude that, depending on the particu- polyglutamine-expanded huntingtin activates Src and lar cellular context, the expanded ataxin-3 can provide a ultimately promotes neuronal death induced by gluta- widespread anti-apoptotic impact on cell signaling. mate. In this line, Src was found to be altered in ataxin-2 patient fbroblasts and ataxin-2 knockout lines [16]. Further supporting our RPMA analysis, we observed Conclusion using Western blots a trend towards reduced phospho- In our work, we used RPMA analysis to characterize rylation of mTOR at Ser2448, an activating modifcation the altered signaling networks observed upon ataxin-3 mediated by AKT and p70S6K [23], and signifcantly expansion in both in vitro and in vivo models. It is increased levels of Ser108-phosphorylated AMPKβ1, important to consider that these models have large dif- which acts as an upstream inhibitor of mTOR [21, 54]. ferences such as innate expression of ataxin-3, overex- Tese observations support the indication of rather pression of ataxin-3, have diferent levels of sensitivity to impaired mTOR activation. In an earlier study, the inhibi- expanded protein and are derived from diferent tissues. tion of mTOR led to an induction of autophagy thereby We employed a set of antibodies which cover various ameliorating the toxicity of expanded ataxin-3 in SCA3 pathways across the cellular landscape including infam- mice [35]. Consistently, increasing phosphorylated mation, survival, energy metabolism, growth, transcrip- AMPK by the administration of cordycepin in SCA3 tion, translation, apoptosis, mitochondrial integrity and cells and mice was shown to have neuroprotective efects autophagy. We suggest that elucidating these signal- via activation of autophagy and lowering mutant pro- ing cascades is integral in understanding the function tein synthesis [32]. Tus, these fndings suggest that the of ataxin-3 in health and disease. We demonstrate that detected lowering of mTOR activation in our models may across our models, AKT/mTOR pathway targets (Table 1) represent a rescue mechanism. are signifcantly altered. Our results revealed alterations shared between tested models as well as the unique dif- Pro‑survival, anti‑apoptotic contribution of polyglutamine ferences providing new perspectives on altered sign- expansion aling in SCA3. In future studies, the role of the altered pathways needs to be correlated to the disease progres- Apoptosis and necrosis are believed to be the two major sion and outcome in SCA3 patients and animal models death pathways for neurons [14]. Regarding apoptosis, [46, 49]. Tis analysis can shed light on future targets for it was suggested that initiator or executor caspases are research and pharmaceutical development by painting a activated in neurodegenerative diseases [57]. In Hunting- clearer picture of the neurodegenerative landscape. ton’s disease, neurons positive for caspase-3 die quickly but for those with aggregates trigger cellular quiescence, deactivate apoptosis but activate delayed necrosis, which Abbreviations SCA3: Spinocerebellar ataxia type 3; MJD: Machado-Joseph disease; AKT: supports the argument that high-ordered aggregates or AKT8 virus oncogene cellular homolog; ATF: Activating transcription factor inclusions might be protective [41]. In our analysis, the 2; Bad: Bcl-2-associated agonist of cell death; Bax: Bcl-2-associated X protein; levels of cleaved caspases including caspase-3, -7, and Bcl-xL: B-cell lymphoma-extra large; Bim: Bcl-2-interacting mediator; cl: Prefx cl- denotes a cleaved form; CREB: CAMP response element-binding -9 were increased in SCA3 mouse brains while either protein; eNOS: Endothelial nitric oxide synthase; ERK: Extracellular signal- decreased or not altered in MEF and HEK293T cell mod- regulated kinase; FADD: Fas-associated protein with death domain; FLIP: FLICE els. Seemingly contradictory behavior was displayed by (Caspase-8) inhibitory protein; FOXO: Forkhead box protein class O4; GAPDH: 148Q Glyceraldehyde-3-phosphate dehydrogenase; GSK: Glycogen synthase kinase; the pro-apoptotic FADD (activated in MEF­ cells, but HSP27: Heat shock protein 27; HSP90: Heat shock protein 90; IκBα: Inhibitor of suppressed in our other models). Bcl-2 family members NF-κB alpha subunit; IL-10: Interleukin 10; JNK: Jun N-terminal kinase; mTOR: (Bcl-xL, Bax, Bad, Bim) as well as FLIP, SHIP and XIAP Mammalian target of rapamycin; NF-κB: Nuclear factor κB; p-: Prefx p- denotes a phosphorylated protein; p53: Tumor suppressor protein that protects from were uniformly either reduced or not altered. Recent data DNA damage; p70S6K: Phosphoprotein 70 ribosomal protein S6 kinase; PARP: showed that the ratio of Bcl2/Bax was decreased in SCA3 Poly(ADP-ribose) polymerase; PTEN: Phosphatase and tensin homolog deleted patients compared to controls, suggesting that the ratios on chromosome 10; RPMA: Reverse-phase Protein MicroArray; RPS6: Small- subunit ribosomal protein S6; SHIP: Src homology 2 (SH2) domain-containing and balances of these proteins are perhaps more impor- inositol phosphatase; Src: Rous sarcoma oncogene cellular homolog; STAT​ tant than their absolute values [61]. Consistent with this, : Signal transducer and activator of transcription; SUMO: Small ubiquitin- comparison of normal vs. expanded ataxin-3 showed like modifer; TNF-R1: Tumor necrosis factor receptor 1; wt: Wild-type; XIAP: X-linked inhibitor of apoptosis.Supplementary Information that the presence of the polyglutamine expansion within ataxin-3 provided a pro-survival and anti-apoptotic con- tribution. Since the cell behavior is dictated by a super- position of multiple signals, the overall balance in favor Sowa et al. Mol Brain (2021) 14:57 Page 9 of 10

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ENOS gene We want to thank Virginia Espina for her technical assistance on this project deletion restores blood-brain barrier integrity and attenuates neuro‑ and Ina Schmitt for providing ataxin-3 knockout mice. We further acknowl‑ degeneration in the thiamine-defcient mouse brain. J Neurochem. edge support by the Open Access Publishing Fund of the Eberhard Karls 2009;111(2):452–9. https://​doi.​org/​10.​1111/j.​1471-​4159.​2009.​06338.x. University of Tuebingen. 7. Bertran-Gonzalez J, Chieng BC, Laurent V, Valjent E, Balleine BW. Striatal cholinergic interneurons display activity-related phosphorylation of Authors’ contributions ribosomal protein S6. PLoS ONE. 2012;7:12. https://​doi.​org/​10.​1371/​journ​ Conceptualization, ASS and TP; Investigation, ASS, TSP, TH, JJW, JS, PSS; Writing- al.​pone.​00531​95. Original Draft, ASS; Writing-Review & Editing, ASS, JHS, JJW, JS, TS; Supervi‑ 8. Beurel E, Jope RS. Diferential regulation of STAT family members by sion, JHS, TS; Funding Acquisition, TS. All authors read and approved the fnal glycogen synthase kinase-3. J Biol Chem. 2008;283(32):21934–44. https://​ manuscript. doi.​org/​10.​1074/​jbc.​M8024​81200. 9. Bichelmeier U, Schmidt T, Hübener J, Boy J, Rüttiger L, Häbig K, Poths S, Funding Bonin M, Knipper M, Schmidt WJ, Wilbertz J, Wolburg H, Laccone F, Riess Open Access funding enabled and organized by Projekt DEAL. The work lead‑ O. Nuclear localization of ataxin-3 is required for the manifestation of ing to this invention has received funding from the European Commission’s symptoms in SCA3: in vivo evidence. J Neurosci. 2007;27(28):7418–28. Seventh Framework Programme FP7/2010 under grant agreement no. 264508 https://​doi.​org/​10.​1523/​JNEUR​OSCI.​4540-​06.​2007. (TreatPolyQ) and the Federal Ministry of Education and Research (PPPT-MJD, 10. Bieging KT, Mello SS, Attardi LD. 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